A. Field Of The Invention
The present invention relates to recombinant DNA technology. It is especially useful in facilitating the introduction of foreign genes into eukaryotic cells.
B. Description Of The Art
There has been much interest in introducing foreign genes into eukaryotic cells. One reason for this interest is that some genetically caused diseases may be curable by introducing the foreign genes into the cells, and allowing the foreign genes to express a protein that the genetically defective cell cannot express. Another reason for this interest is that certain eukaryotic cells may prove to be the most suitable hosts for the production of certain eukaryotic proteins. Yet another reason is that this may permit farm animals to be genetically improved.
A promising approach for introducing foreign DNA into eukaryotic cells involves the use of retrovirus vectors. See J. Dougherty et al., 84 P.N.A.S. U.S.A. 1197-1201 (1987); M. Eglites et al., 6 BioTechniques 608-614 (1988). Retroviruses integrate into a target cell's chromosomes during their life cycle. By modifying the virus genome to include a gene of interest one has a means of carrying desired genes into the cell chromosomes. As a safety feature, the genetic material of the retrovirus vector is further modified so as to make it defective in virion structural proteins (e.g. gag, pol, env). This reduces the likelihood of the retrovirus replicating after integration in the target cell.
Helper (a.k.a. packaging) cell lines therefore had to be developed to permit the propagation of replication-defective retrovirus vectors. These cells operate by supplying proteins that the defective vector cannot supply for itself. Preferably, such helper cells are designed so as to produce the vectors without the presence of live helper viruses. See e.g. U.S. Pat. No. 4,650,764. In the U.S. Pat. No. 4,650,764 system, the helper sequences provide virion proteins (e.g. env, gag, pol) that the vectors have been designed not to have. The helper sequences are also defective (albeit in a different way, in encapsidation function).
A problem has arisen in using such helper cells. On occasion, spontaneous recombination in the helper cell of vector sequences with helper sequences leads to the formation of sequences that code for replication-competent retroviruses. One early attempt to reduce this recombination risk is described in U.S. Pat. No. 4,650,764. The reticuloendotheliosis virus gag-pol and env genes were positioned so as to be expressed from two separate segments. While this approach helped somewhat, this improvement alone was not sufficient to eliminate all problems.
Another problem with the use of helper cells was that they would not permit standard vectors to be efficiently used beyond a relatively limited host range.
Therefore, improved helper cells for use with retrovirus vectors are desired.